INSTRUMENTAL ANALYSIS CHEM 4811

CHAPTER 13

DR. AUGUSTINE OFORI AGYEMANAssistant professor of chemistryDepartment of natural sciences

Clayton state university

CHAPTER 13

LIQUID CHROMATOGRAPHY (LC)

LC STATIONARY PHASE

- Usually silica (SiO2 · xH2O)

- Alumina (Al2O3 · xH2O)

- Adsorption of water slowly deactivates adsorption sites of silica

- May be reactivated by heating to about 200oC in an oven

ELUENT STRENGTH (εo)

- A measure of adsorption energy of solvent

- εo of pentane is 0

- More polar solvents have greater eluent strengths

- Solutes elute more rapidly when eluent strength is greater

- Weakly retained solutes are first eluted with alow eluent strength solvent

- Eluent strength is increased by adding a polar solvent to elute strongly retained solutes

- Eluent strength is increased by making mobile phase more like the stationary phase

Increasing order of polarity contributionHCs < ethers < esters < ketones < aldehydes < amides

< amines < alcohols

ELUENT STRENGTH (εo)

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

- Good for Proteomics (analysis of amino acids and peptides)

- Uses closed packed columns under high pressure

- Resolution increases with decreasing particle size of stationary phase

- Decreasing particle size decreases plate height

- Small particle size reduces the terms A and C in the van Deemter equation

HPLC STATIONARY PHASE

- Microporous spherical particles of silica (2 – 10 µm)

- Long chain C18 hydrocarbon bonded to silica surface (Octadecyl)

- Particles must be stable, uniform, spherical for uniform flow

- The area covered by stationary phase is greater than that of the polymer film in GC

- Column operates at very high pressures (500 – 6000 psi)

HPLC STATIONARY PHASE

- The organosilyl bonds may hydrolyze at very low pH

- Silica may dissolve at high pH (above 8)

- Silica can become soluble at very high temperatures

- Zirconia (ZrO2) is used as particle support which has greater pH range stability (1 – 14) and temperature stability (> 100 oC)

- Cross-linked styrene-divinylbenzene copolymer (called XAD resins) are also used

NORMAL PHASE HPLC

- Stationary phase is polar

- Mobile phase is less polar

- Eluent strength is increased by adding a more polar solvent

Examples of organic substituents for stationary phaseAmino [–(CH2)3NH2] Cyano [–(CH2)3–C≡N]

Diol [–(CH2)2OCH2CH(OH)CH2OH]

- More common

- Stationary phase is weakly polar or nonpolar

- Mobile is more aqueous or polar

- Insensitive to polar impurities and eliminates tailing

Examples of organic substituents for stationary phaseOctadecyl [–(CH2)17CH3; C18,ODS]

Octyl [–(CH2)7CH3; C8] Phenyl [–(CH2)3C6H5]

REVERSE PHASE HPLC (RP-HPLC)

- Mobile Phase Degassing (reservoirs with He gas for removal of dissolved gases)

- Mobile Phase Storage (reservoirs for storing mobile phase)

- Mobile Phase Mixing (for mixing solvents from reservoirs)

- HPLC Pump (generates high pressure for high performance)

HPLC INSTRUMENTATION

- Fill/Drain Valve (liquid transfer lines and components)

- Rotary Sample Loop Injector (introduces sample with syringe)

- The Column- Irreversible adsorption to the columns is very common

- Disposable guard column containing the same stationary phase is attached to the column entrance

- HPLC Detectors

HPLC INSTRUMENTATION

Isocratic Elution- Elution with a single solvent

- Elution with a constant solvent mixture

Gradient Elution- Solvent is changed continuously from weak to

strong eluent strength

- Used when the solvent does not efficiently elute all components

HPLC INSTRUMENTATION

HPLC DETECTORS

Refractive Index (RI) Detector- Universal, nondestructive, concentration detector

- Relatively insensitive and for only isocratic separations

Evaporative Light Scattering Detector (ELSD)- Universal, destructive, mass-flow detector

- Sensitive and for isocratic or gradient separations

UV-VIS Absorption Detectors (most common)- Nondestructive, concentration detector

- Varying sensitivity and for isocratic or gradient separations

HPLC DETECTORS

Fluorescence Detector- Compound specific, nondestructive, concentration detector- Varying sensitivity and for isocratic or gradient separations

Electrochemical Detectors (ECD)- Compound specific, destructive, concentration or mass-flow

- Varying sensitivity and for isocratic RP-HPLC

Conductometric Detector-For cations and anions in solution, nondestructive, concentration

- For isocratic RP-HPLC and ion chromatography

Charged Aerosol Detector (most sensitive)

HPLC HYPHENATED TECHNIQUES

HPLC-UV-VIS

LC-NMR

HPLC-MS (THERMOSPRAY INTERFACE)

HPLC-MS (ELECTROSPRAY INTERFACE)

HPLC-MS (APCI INTERFACE)

ION-EXCHANGE CHROMATOGRAPHY

- Allows separation of ions and polar molecules

- Ionic groups are covalently attached to a stationary solid phase

- Mobile phase is a liquid

- Ionic solutes are electrostatically attracted to the stationary phase

AFFINITY CHROMATOGRAPHY

- Very selective

Powerful for separating a single biomolecule from a complex mixture of biomolecules

- Based on specific interactions between a type of solute molecule and another molecule covalently

attached to the stationary phase

SIZE EXCLUSION CHROMATOGRAPHY

- Also known as Gel Filtration or Gel Permeation

- Solutes are separated based on size

- Stationary phase has small pores that exclude large molecules

- Small molecules enter the pores so spend more time in column

- Large molecules come out of column before small molecules

ADSORPTION CHROMATOGRAPHY

- Stationary phase is a solid

- Mobile phase is a liquid or a gas

- Solute adsorbs to the surface of the solid particles

PARTITION CHROMATOGRAPHY

- Stationary phase is a thin liquid coated on the surfaceof a solid support

- Mobile phase is a liquid or a gas

- Solute equilibrates between the stationary and mobile phases

SUPERCRITICAL FLUIDCHROMATOGRAPHY (SFC)

- Applies the ability of supercritical fluids to dissolve poorly volatile molecules

- Stationary phase is commonly used column packings

- Mobile phase is a supercritical fluid (CO2, C2H6, N2O)

- For extracting caffeine from coffee beans and nicotine from cigarette tobacco

- Employs chiral phases to separate enantiomers

- Cyclodextrins (complex cyclic carbohydrates) bind enantiomers at different strengths enabling separation

- The cyclodextrin pockets come in different sizes which is suitable for separation of enantiomer pairs of different sizes

- Relative amount of each enantiomer can also be determined

- Chiral GC columns are also available

CHIRAL CHROMATOGRAPHY

ELECTROPHORESIS

- Migration of ions in an electric field

- Cations are attracted to the negative electrode (cathode)

- Anions are attracted to the positive electrode (anode)

ELECTROOSMOSIS

- The application of electric field to drive fluid in a capillary tube from anode towards cathode

Capillary Electrophoresis (CE) - Is a combination of electrophoresis and electroosmosis

CAPILLARY ELECTROPHORESIS(CE)

- High-resolution separation method

- Separates charged and neutral analytes in a narrow capillary tube

- Capillary tube wall is usually fused silica

Good technique for the separation of- Small ions (Na+, K+)

- Proteins- DNA

- A buffer solution in the electrode reservoirs

- Controls pH and ionic strength

CE BACKGROUND ELECTROLYTE (RUN BUFFER)

- Both ends of the capillary tube is first immersed in a background electrolyte

- One end of tube is dipped in vial containing the sample

- Pressure or electric field is applied to introduce ~10 nLof sample into the capillary

- Capillary is placed back into the electrolyte

- 20 to 30 kV is applied and causes ions in capillary to migrate

PRINCIPLES OF CE

- Different ions migrate at different speeds

- This results in separation of ions

- Ions reach a detector and an electropherogram (response versus time) is recorded

- Very narrow bands are usually seen

- Terms A and C in the van Deemter equation are reduced(no multiple paths and no stationary phase)

PRINCIPLES OF CE

- Electroosmosis sweeps analyte molecules towards the detector

- Detector is placed near the cathode

- Detector is set to a wavelength of about 200 nm

- Cations reach the detector first

- Neutral molecules reach the detector after cations

- Anions reach the detector after neutral molecules

PRINCIPLES OF CE

At High pH- Electroosmosis is faster than electrophoresis

- Net flow of anions is towards the cathode

At Neutral pH- Electroosmosis is faster than electrophoresis

- Net flow of anions is towards the cathode

PRINCIPLES OF CE

At Low pH- Electroosmosis is slower than electrophoresis

- Net flow of anions is towards the anode

- Anions may not reach the detector

- Polarity can be reversed to separate anions

PRINCIPLES OF CE

Capillary Ozone Electrophoresis (CZE)- Separation is based on different velocities of different ions

Capillary Gel Electrophoresis (CGE)- Separation is based on size of molecules

- Molecules are separated upon migrating through a gel in the capillary column

- The process is called sieving- Small molecules travel faster than large molecules

MODES OF CE

Cpillary Isoelectric Focusing (CIEF)- Used separate proteins, peptides, amino acids

- Basis is the presence of both the acidic and basic groups- Separated is based on different isoelectric points by varying pH- Compounds exist as zwitterion with no net charge at a given pH

Micellar Electrokinetic Capillary Chromatography (MEKC)- Used to separate different neutral molecules

- Also used for ions- Micelles are added to the background electrolyte

- An example is sodium dodecyl sulfate (a surfactant)

MODES OF CE

Similar to those of HPLC

- Ultraviolet absorption (most common)

- Conductivity

- Electrochemical

- Fluorescence

DETECTORS